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Read alignment

Learning outcomes

After having completed this chapter you will be able to:

  • Explain what a sequence aligner does
  • Explain why in some cases the aligner needs to be ‘splice-aware’
  • Calculate mapping quality out of the probability that a mapping position is wrong
  • Build an index of the reference and perform an alignment of paired-end reads with bowtie2

Material

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Exercises

Prepare the reference sequence

Retrieve the reference sequence using esearch and efetch:

REFERENCE_DIR=~/workdir/ref_genome/

mkdir $REFERENCE_DIR
cd $REFERENCE_DIR

esearch -db nuccore -query 'U00096' \
| efetch -format fasta > ecoli-strK12-MG1655.fasta

Exercise: Check out the documentation of bowtie2-build, and build the indexed reference genome with bowtie2 using default options.

Answer
bowtie2-build ecoli-strK12-MG1655.fasta ecoli-strK12-MG1655.fasta

Align the reads with bowtie2

Exercise: Check out the bowtie2 manual here. We are going to align the sequences in paired-end mode. What are the options we’ll minimally need?

Answer

According to the usage of bowtie2: 

bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r> | --interleaved <i> | --sra-acc <acc> | b <bam>}

We’ll need the options:

  • -x to point to our index
  • -1 and -2 to point to our forward and reverse reads

Exercise: Try to understand what the script below does, and run it.

##!/usr/bin/env bash

TRIMMED_DIR=~/workdir/trimmed_data
REFERENCE_DIR=~/workdir/ref_genome/
ALIGNED_DIR=~/workdir/alignment_output

mkdir $ALIGNED_DIR

bowtie2 \
-x $REFERENCE_DIR/ecoli-strK12-MG1655.fasta \
-1 $TRIMMED_DIR/paired_trimmed_SRR519926_1.fastq \
-2 $TRIMMED_DIR/paired_trimmed_SRR519926_2.fastq \
> $ALIGNED_DIR/SRR519926.sam

We’ll go deeper into alignment statistics later on, but bowtie2 writes already some statistics to stdout. General alignment rates seem okay, but there are quite some non-concordant alignments. That doesn’t sound good. Check out the explanation about concordance at the bowtie2 manual. Can you guess what the reason could be?